We report mutations of the type II
3 beta-hydroxysteroid dehydrogenase (3 beta HSD) gene in two siblings, male and female, with
congenital adrenal hyperplasia caused by classical nonsalt-losing
3 beta HSD deficiency. During childhood, the male sibling, born with
ambiguous genitalia, and the female sibling, born with normal genitalia, both manifested symptoms of mild
androgen excess; both apparently had normal zona glomerulosa function. Gonadal dynamic study at puberty showed the presence of partial gonadal
3 beta HSD deficiency in both siblings despite their spontaneous pubertal maturation. The 5'-region as well as exons I-II, III, and IV and portions of the adjacent introns of the type II 3 beta HSD gene were amplified by polymerase chain reaction and sequenced. In both siblings and their mother, an identical single
nucleotide substitution mutation in intron III, six bases up-stream from exon IV, was identified in one allele. This mutation, G to A at
nucleotide 6651, may create a new splicing junction and affect the normal splicing of the messenger
ribonucleic acid. In the other allele of both siblings, a missense mutation from GGG (Gly) to AGG (Arg) at
codon 129 (G129R) in exon IV was found. We assessed the effect of the G129R missense mutation on enzymatic activity by in vitro analysis of the mutant recombinant
enzyme generated by site-directed mutagenesis after its transient expression in COS-1 cells. Using homogenates from transfected cells, the G129R 3 beta HSD
enzyme showed a Km value for
pregnenolone of 10 +/- 2 mumol/L compared with 1.00 +/- 0.03 mumol/L for the wild-type type II 3 beta HSD
enzyme. When
dehydroepiandrosterone was used as substrate, the Km value for G129R3 beta HSD was 14 +/- 2 mumol/L compared with 2.1 +/- 0.2 mumol/L for the wild-type II 3 beta HSD
enzyme. In addition to an apparent decrease in affinity, the G129R mutation caused a marked decrease in the apparent relative specific activity, thus leading to apparent relative specific efficiencies (relative specific activity/Km) of 2.0% and 4.7% that of the normal type II 3 beta HSD using
pregnenolone or
dehydroepiandrosterone as substrate, respectively. It appears likely that this low level of activity is sufficient to prevent
salt loss, but it is also possible that part of the enzymatic activity comes from the putative remaining percentage of correctly spliced n6651 allele in these patients.